Anion Binding by Human Lactoferrin: Results from Crystallographic and Physicochemical Studies

The anion-binding properties of lactoferrin (Lf), with Fe³⁺ or Cu²⁺ as the associated metal ion, have been investigated by physicochemical and crystallographic techniques. These highlight differences between the two sites and in the anion-binding behavior when different metals are bound. Carbonate, oxalate, and hybrid carbonate-oxalate complexes have been prepared and their characteristic electronic and EPR spectra recorded. Oxalate can displace carbonate from either one or both anion sites of Cu₂(CO₃)₂Lf, depending on the oxalate concentration, but no such displacement occurs for Fe₂(C0₃)₂Lf. Addition of oxalate and the appropriate metal ion to apoLf under carbonate-free conditions gives dioxalate complexes with both Fe³⁺ and Cu²⁺, except when traces of EDTA remain associated with the protein, when hybrid complexes M₂(CO₃)(C₂0₄)Lf can result. The anion sites in the crystal structures of Fe₂(CO₃)₂Lf, Cu₂-(C0₃)₂Lf, and Cu₂(CO₃)(C₂O₄)Lf, refined at 2.2, 2.1, and 2.2 Å, respectively, have been compared. In every case, the anion is hydrogen bonded to the N-terminus of helix 5, an associated arginine side chain, and a nearby threonine side chain. The carbonate ion binds in bidentate fashion to the metal, except in the N-lobe site of dicupric lactoferrin, where it is monodentate; the difference arises from slight movement of the metal ion. The hybrid complex shows that the oxalate ion binds preferentially in the C-lobe site, in 1, 2-bidentate mode, but with the displacement of several nearby side chains. These observations lead to a generalized model for synergistic anion binding by transferrins.